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Objective: To evaluate HPV16 CpG methylation and methyl-haplotypes and their association with cervix precancer and cancer utilizing massively parallel single molecule next-generation sequencing (NGS)., Methods: A nested case-control study of HPV16 positive women was performed in a prospective cohort from Guanacaste, Costa Rica designed to study the natural history of HPV and cervical neoplasia. Controls encompassed 31 women with transient infections; there were 44 cases, including 31 women with CIN3 and 13 with cervical cancer. DNA samples from exfoliated cervical cells were treated with bisulfite and four regions (E6, E2, L2 and L1) were amplified with barcoded primers and tested by NGS. CpG methylation was quantified using a bioinformatics pipeline., Results: Median methylation levels were significantly different between the CIN3+ cases versus controls in the E2, L2, and L1 regions. Methyl-haplotypes, specifically in 5 CpG sites included in the targeted L2 region, with the pattern "--+-+" had the highest Area Under the Curve value (AUC=88.40%) observed for CIN3 vs., Controls: The most significant CpG site, L2 4277, determined by bisulfite NGS had an AUC=78.62%., Conclusions: This study demonstrates that NGS of bisulfite treated HPV DNA is a useful and efficient technique to survey methylation patterns in HPV16. This procedure provides quantitative information on both individual CpG sites and methyl-haplotypes that identify women with elevated present or subsequent risk for HPV16 CIN3 and cancer., (Copyright © 2015 Elsevier Inc. All rights reserved.)